Isolating and cryopreserving pig skin cells for single-cell RNA sequencing study

The pig skin architecture and physiology are similar to those of humans. Thus, the pig model is very valuable for studying skin biology and testing therapeutics. The single-cell RNA sequencing (scRNA-seq) technology allows quantitatively analyzing cell types, compositions, states, signaling, and receptor-ligand interactome at single-cell resolution and at high throughput. scRNA-seq has been used to study mouse and human skins. However, studying pig skin with scRNA-seq is still rare. A critical step for successful scRNA-seq is to obtain high-quality single cells from the pig skin tissue. Here we report a robust method for isolating and cryopreserving pig skin single cells for scRNA-seq. We showed that pig skin could be efficiently dissociated into single cells with high cell viability using the Miltenyi Human Whole Skin Dissociation kit and the Miltenyi gentleMACS Dissociator. Furthermore, the obtained single cells could be cryopreserved using 90% FBS + 10% DMSO without causing additional cell death, cell aggregation, or changes in gene expression profiles. Using the developed protocol, we were able to identify all the major skin cell types. The protocol and results from this study are valuable for the skin research scientific community.

In a typical scRNA-seq workflow, tissues are first dissociated into single cells (i.e., the upstream single cell preparation). These freshly isolated cells are then used to prepare RNA libraries using droplet-based technology (i.e., the downstream library preparation) [31]. The libraries are then sequenced using the deep sequencing technology [32]. Preparing high-quality single cells is critical for the success of the downstream process. Partial or incomplete tissue dissociation, cell aggregation, and cell death should be avoided during the preparation.
Recently studies showed that adding a cell preservation step between the upstream single cell preparation and downstream RNA library construction makes the scRNA-seq workflow much more flexible, manageable, and accessible to researchers [24,[33][34][35][36]. First, some research facilities/institutions do not have the infrastructure and expertise to prepare scRNA-seq libraries for the freshly isolated cells. Instead, they ship cells to service facilities for RNA library construction and sequencing. Cells should be preserved during shipping. Second, most studies collect samples at multiple time points and different locations. Preparing RNA libraries at the same facilities can increase consistency and reduce experimental variations. To achieve this, cells need preservation. A suitable cell preservation method should not significantly change the cell viability, composition, and gene expression [24,[33][34][35][36]. To our best knowledge, there have been no reports on how to prepare and preserve single cells from pig skin tissues for scRNA-Seq, which this study aims to address.

Harvesting pig skin
Fresh skin tissues from healthy farm male pigs with about 30 kg of body weight were provided by the University of Nebraska-Lincoln Swine Facility. The Institutional Animal Care and Use Committee of the University of Nebraska-Lincoln granted a waiver of ethics approval for using skins harvested from dead animals. The dorsal area of the skin was washed with PBS, and the fur was removed with a disposable scalpel. The skin was disinfected with 70% ethanol, harvested using sterile scissors, and stored in the MACS Tissue Storage Solution (Miltenyi Biotech Inc) with 1% Antibiotic-Antimycotic (ThermoFisher Scientific). The samples were kept on ice and transported to the lab for single cell isolation.

Isolating single cells
The skin was washed with sterile iced cold PBS three times, and the subcutaneous fat was scraped off using a scalpel. A full skin sample, including the epidermis and dermis, was taken with a 4-mm diameter punch. The sample was then dissociated using the Human Whole Skin Dissociation Kit (Miltenyi Biotech Inc). Briefly, 435 μL of Buffer L and 12.5 μL of Enzyme P were put into a gentleMACS C tube and gently mixed before adding 50 μL of Enzyme D and 2.5 μL of Enzyme A. One skin sample was placed into the tube and incubated at 37˚C for three hours. The tube was inverted for mixing every ten minutes. The enzymatic reaction was stopped by adding 0.5 mL ice-cold cell culture medium (DMEM + 10%FBS). The sample was then mechanically dissociated with a gentleMACS Dissociator (Miltenyi Biotech Inc) using the "h_skin_01" program. The suspension was passed through a 70 μm strainer to remove tissue debris, if any. Cells were collected by centrifugation at 300 g for 5 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 5 mL DMEM + 10% FBS. Cells were counted using a Countess II FL Automated Cell Counter (ThermoFisher Scientific) on a 0.2% trypan blue staining. The viability was further assessed using the LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (Invitrogen).

Cryopreserving cells
The freshly isolated cell suspension was centrifuged at 300 g for 5 minutes. The cell pellet was resuspended with FBS containing 10% DMSO at 1x10 6 cells/mL. Next, 1 mL cells suspension was put in a cryopreservation vial and placed in a Mr. Frosty Freezing Container (Thermo-Fisher Scientific). The container was placed in a -80˚C freezer overnight before being stored in liquid N 2 for the long term.

Thawing cells
The frozen vial was removed from the liquid N 2 storage and placed in a 37˚C water bath. After thawing, the cell suspension was centrifuged at 300 g for 5 minutes and resuspended in DMEM + 10% FBS. The cell viability was assessed with the LIVE/DEAD™ Viability/Cytotoxicity Kit and flow cytometer.

Removing dead cells using Magnetic-Activated Cell Sorting (MACS)
An alternative approach was used to remove dead cells with the Dead Cell Remove Kit (Miltenyi Biotech Inc) following the product instruction. Briefly, the cell suspension was centrifuged at 300 g for 5 minutes. Cells were resuspended in 100 μL of Dead Cell Removal MicroBeads and incubated for 15 minutes at room temperature. Dead cells were removed using a MACS Separator (Miltenyi Biotech Inc) following the product instruction.

Library construction
Cells were suspended in DMEM+10% FBS. The cells' density and viability were estimated using a Countess II FL Automated Cell Counter. About 8,000 cells were used as the input to generate an RNA-seq library following the 10X Genomics Chromium Next GEM Single Cell 3 0 kit V3 protocol. Libraries were sequenced at Novogene using NovaSeq 6000 sequencer.

Data processing
scRNA-seq data was processed with Cell Ranger pipeline version 4.0.0. (10x Genomics). A reference transcriptome was created by utilizing the pig reference genome (Sscrofa11.1) and its annotation downloaded from Ensemble (https://www.ensembl.org/Sus_scrofa/Info/Index). The reads were mapped using STAR aligner (v. 2.5.1b) against the reference transcriptome to detect and count UMIs and expressed genes.

Data analysis
The quality of sequencing data was assessed using quality control parameters, including the gene counts per cell, UMI counts per cell, and mitochondrial gene expression [37,38]. These quality control parameters were calculated as part of the scRNA-seq data analysis procedure using the Seurat R package version 3.2.1 [37]. Since cell doublets or multiplets exhibit an aberrantly high gene and molecule counts, we set a maximum threshold at 4,500 genes per cell and 30,000 molecules per cell (S1 Fig). Also, we allowed cells up to 10% mitochondrial gene expression (S1 Fig).
The sample data sets were merged into one R Data object per experiment for joint cluster analysis. Then, the UMI count data per cell were normalized and log-transformed using the default settings of the "Normalize Data" function in Seurat [37]. Principal component analysis (PCA) was performed using the highly variable genes for each sample. Significant principal components were selected for subsequent cluster analysis. Single cell clustering was visualized with uniform manifold approximation and projection (UMAP) plots with default parameters. Cell types were annotated using marker genes with at least a 2-fold increase in individual cell clusters compared to the remaining cells. We used CellMatch for automated cell type annotation [39]. Based on the evidence-based score, we annotated the clusters by matching the identified marker genes with known cell markers in tissue-specific cell taxonomy reference databases [39]. For single-cell trajectory analysis [40,41], we used an algorithm to learn the changes in each cell's gene expression sequence. Once the algorithm has learned the overall "trajectory" of gene expression changes, we placed each cell at its proper position in the trajectory (line).
Statistically significant differences between cell cluster gene levels were calculated using the MAST linear model approach, as implemented in the Seurat package. Genes were considered as being significantly altered if gene expression levels had at least 2-fold changes and adjusted P-values were less than 0.001 (Bonferroni correction). The overall similarity of fresh and cryopreserved samples' gene expression profiles was assessed by the correlation performed on "pseudo-bulk" expression profiles [42], which were generated by summing counts together for all cells within the same sample by using the function of "aggregateAcross" in the Scatter package [43]. The raw pseudo-bulk count matrices were normalized using edgeR version 3.30.3 [44]. Pearson correlation of the fresh and cryopreserved samples was computed using the normalized counts. The differentially expressed pseudo-bulk genes were identified by edgeR.

Statistical analysis
The data are presented as the mean ± S.D. We used an unpaired t-test to compare two groups and one-way ANOVA to compare more than two groups. P-value < 0.05 was considered statistically significant. We used GraphPad Prism 6 for Windows 6.01v to perform statistical analysis.

Isolating and preserving single pig skin cells
We used the Miltenyi Human Whole Skin Dissociation Kit and the gentleMACS Dissociator to dissociate the pig skin. Our results showed the combination efficiently dissociated the skin into single cells with only a few cell aggregates and no cell or tissue debris. The resultant cells had healthy and spherical morphology (Fig 1A and 1B). Live/dead cell staining showed that most cells were live, and confirmed cell aggregates were few (Fig 1C and 1D). Flow cytometry analysis showed that >60% of cells were viable (Fig 1E). We froze the freshly isolated cells in 90% FBS + 10% DMSO at -80˚C, followed by long-term storage in liquid N 2 . After thawing, cryopreserved cells had similar spherical morphology and viability as the fresh cells (Fig 1A-1E), preliminarily indicating this method is appropriate for preserving cells. Furthermore, the cryopreservation did not induce cell aggregation.
Cell aggregates interfere with the downstream library preparation. RNAs released from dead cells negatively affect scRNA-seq results. We sought to remove both aggregated and dead cells with fluorescence-activated cell sorting (FACS) before the library preparation. Dead cells were stained with ethidium homodimer-1 dye and removed via the red fluorescence. Cell doublets were removed using the side-scatter and forward-scatter profiles. FACS quantitively removed both (Fig 1D). Since many researchers have difficulty accessing a FACS instrument, we also used an alternative and compact device, the Miltenyi magnetic-activated cell sorting (MACS), to remove the dead cells. MACS quantitively removed dead cells. However, it was not efficient to remove cell aggregates (Fig 1D). In summary, it is appropriate to combine the

PLOS ONE
Human Whole Skin Dissociation Kit, the gentleMACS Dissociator, cryopreservation, and FACS to prepare and preserve high-quality single pig skin cells for scRNA-seq.

Cryopreservation in 90% FBS + 10% DMSO preserved gene expression
Our data showed the genes per cell, UMIs per cell, % mitochondrial genes were similar between the fresh and the cryopreserved sample (Fig 2A-2C). The pseudo-bulk expression profiles of cryopreserved and fresh cells were compared using correlation analysis to assess if cryopreservation alters the gene expression. The expression profile of the cryopreserved sample correlated very well with this of fresh cells (R = 0.981) (Fig 3A). Among 20,428 expressed genes, there were only 57 differentially expressed genes (DEGs) between fresh and cryopreserved samples with FDR adjusted P-values < 0.01. This indicates the gene expression profiles between fresh and cryopreserved samples are almost the same. Next, we analyzed the subset of genes that exhibited high cell-to-cell variation in the samples (Fig 3B). Fresh and cryopreserved samples shared the highly variable features (e.g., CD209, MMP3, CCL5, C4BPA) with similar variance and average expression. Lastly, we assembled the fresh and the cryopreserved sample into an integrated reference and visualized the integration using a non-linear dimensional reduction UMAP (Fig 3C). Again, the fresh and cryopreserved cells formed similar structures. These results show that cryopreservation in 90% FBS + 10% DMSO did not alter the gene expression significantly.

PLOS ONE
Single cell RNA sequencing study of pig skin cells

Cryopreservation in 90% FBS + 10% DMSO retained the major skin cell types
Next, we sought to answer if the single cell preparation protocol could retain the major skin cell types and if the cryopreservation changed the cell composition. We performed a clustering analysis of the integrated cryopreserved and fresh cells using a graph-based clustering approach [37,45]-the Louvain algorithm [46]. This method embeds cells in a graph structure, clusters cells with similar feature expressions, and partitions the graph into highly interconnected communities. We identified 18 clusters (S2 Fig) and the top 10 marker genes for each cluster (S3 Fig). We then used a mixed strategy to annotate the clusters. We used CellMatch [46] to annotate clusters based on the evidence-based score by matching the identified marker genes with known cell markers in tissue-specific cell taxonomy reference databases. Also, we used cell marker genes from recent scRNA-seq studies of skin [16,27] to identify the remaining cell types. This allows us to identify the major skin cell types (Fig 4A). The expression levels for the markers of each cell type were shown in Fig 5 and S1 Table. The top 10 marker genes for each cell type were also identified (S4 Fig). We compared the fresh and the cryopreserved samples regarding the relative organization and connection between cell clusters (Fig 4A). The two

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Single cell RNA sequencing study of pig skin cells samples were very similar. Also, the cryopreservation did not significantly alter the cell compositions (Table 1).

Cryopreservation retained the cell cycle status and differentiation trajectory
Further, we used cell cycle regression to analyze the effect of cryopreservation on the cell cycle status. We identified the cell cycle heterogeneity from cryopreserved and fresh samples by calculating cell cycle phase scores based on canonical markers [39]. Our results showed no differences between fresh and cryopreserved keratinocytes regarding G2/M-phase genes (Fig 4B).
Using the single-cell trajectory analysis [40,41], we found that epidermal stem cells differentiated into keratinocytes in both fresh and cryopreserved samples (Fig 6A and 6B). The results indicate the data quality for both the fresh and cryopreserved cells is sufficient for transcriptional trajectory analysis.

Discussion
Preparing high-quality single cells is a critical step for a successful scRNA-seq study. A suitable dissociation method should dissolve the whole skin tissue, including epidermis and dermis, to release most if not all cells [23,47,48]. The resultant cells should have high viability and few aggregates. A dissociation method with high cell yield and cell viability can minimize skin samples and allow detecting cell types with small numbers. A few protocols have been published for preparing single cells from rodent [49][50][51][52][53][54][55] and human skins [56][57][58][59][60][61] via enzymatic digestion and mechanical dissociation. These methods vary in enzyme types and concentrations, digestion time, and temperature [49][50][51][52][53][54][55][56][57][58][59][60][61]. Most of them use manual mechanical dissociation, such as dissection, mincing, agitation, pipetting, or passing through syringes that are inefficient and inconsistent. Consequently, the dissociation efficiency and the resultant cell viability, composition vary between methods, publications, and batches. The variations create challenges when comparing scRNA-seq results from different labs. Thus, there is a critical need to develop a standardized single cell preparation method. A standard procedure could be best achieved using validated, commercially available enzyme kits and automated mechanical dissociation devices. Although we could not find a commercial kit designed explicitly for pig skin, the Miltenyi Human Whole Skin Dissociation Kit and the gentleMACS Dissociator had been used to prepare human skin single cells for scRNA-seq [23,24,27,28,62,63]. Due to the similarity of the human and pig skins, we hypothesized that they could also be used to prepare single pig skin cells. With our protocol, the skin tissue was close to completely dissolved. Only small numbers of cell aggregates were found, and they could be robustly removed with FACS (Fig 1). We were able to isolate 6.0-7.5×10 4 viable single cells with a 4-mm full skin biopsy punch. These cells are sufficient for a complete scRNA-seq flow, including cell counting, cell viability, quality assessment, RNA library construction, and quality control.
Reliable methods for preserving single cells make the scRNA-Seq workflow much more flexible and manageable [24,[33][34][35][36]. A few methods, including cryopreservation in medium containing DMSO, using commercial cell preservation reagent, or methanol fixing, have been reported to preserve single cells [24,33,35,36]. DMSO minimizes forming large intracellular ice crystals, which damage and kill cells. Methanol fixing works through dehydrating cells to preserve nucleic acids in a collapsed form at high concentrations. Upon rehydration, nucleic acids can be restored to their original form and harvested for library preparation [62,64]. Literature research showed the methanol fixing method result in high ambient RNA background and a lower gene expression correlation to un-preserved cells [33]. The same was true for when using commercial CellCover reagent [33]. On the other hand, cryopreserving human and rodent primary cells or cell lines using DMSO plus FBS did not reduce the cell viability and alter the cell composition and gene expression significantly [24,[33][34][35][36]65]. Our results with pig skin cells (Figs 1-6) agreed well with these literature studies.
In summary, we showed that high-quality single pig skin cells could be generated using the Miltenyi Biotec Whole Skin Dissociation kit and Gentle MACS Dissociator. Single cells could be cryopreserved in 90% FBS+10% DMSO, and the cryopreservation did not significantly alter gene expression and cell compositions. Dead cells and cell aggregates could be removed via FACS before preparing libraries. Using these methods, we obtained high-quality scRNA-Seq data to identify the major skin cell types. The limitation of this study is that only healthy pig skins from young pigs were used. Future studies should test if the protocol works for aged or diseased pig skins.